Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen.

CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia.

Disease relapse or progression is a major cause of death following umbilical cord blood (UCB) transplantation (UCBT) in patients with high-risk, relapsed or refractory acute lymphoblastic leukemia (ALL). Adoptive transfer of donor-derived T cells modified to express a tumor-targeted chimeric antigen receptor (CAR) may eradicate persistent disease after transplantation. Such therapy has not been available to UCBT recipients, however, due to the low numbers of available UCB T cells and the limited capacity for ex vivo expansion of cytolytic cells. We have developed a novel strategy to expand UCB T cells to clinically relevant numbers in the context of exogenous cytokines. UCB-derived T cells cultured with interleukin (IL)-12 and IL-15 generated >150-fold expansion with a unique central memory/effector phenotype. Moreover, UCB T cells were modified to both express the CD19-specific CAR, 1928z, and secrete IL-12. 1928z/IL-12 UCB T cells retained a central memory-effector phenotype and had increased antitumor efficacy in vitro. Furthermore, adoptive transfer of 1928z/IL-12 UCB T cells resulted in significantly enhanced survival of CD19(+) tumor-bearing SCID-Beige mice. Clinical translation of CAR-modified UCB T cells could augment the graft-versus-leukemia effect after UCBT and thus further improve disease-free survival of transplant patients with B-cell ALL.

Development of spontaneous factor VIII inhibitor in association with acute graft-versus-host disease.

A 57-year-old female with recurrent AML underwent a T cell-depleted (TCD) bone marrow (BM) plus TCD and CD34-selected peripheral blood stem cell (PBSC) transplant. Eleven weeks post transplantation, the patient developed acute graft-versus-host disease (GVHD) manifested by rash and elevated liver enzymes. Concurrently, the patient presented with a bleeding diathesis and a left forearm hematoma due to the development of a spontaneous factor VIII inhibitor. She was treated with human recombinant factor VIII and intravenous methylprednisolone. Subsequently she was managed with a prednisone taper leading to resolution of the GVHD, as well as the spontaneous factor VIII inhibitor. Bone marrow transplant-related spontaneous factor VIII inhibitor has previously been reported in association with one patient with chronic GVHD. To our knowledge this is the first report of spontaneous factor VIII inhibitor associated with acute GVHD.

Islet cell tumors of the pancreas: the medical oncologist's perspective.

Islet cell tumors of the pancreas are rare, indolent, neuroendocrine tumors. Approximately 50% of the patients diagnosed with these tumors present with symptoms related to various biologically active hormones that are secreted by these neoplasms. Currently, the only curative treatment for islet cell tumors is complete surgical resection. Management of metastatic disease is conservative. Initial treatment of these tumors includes expectant observation and medical management of symptoms with clinical monitoring and serial CT scans to assess tumor growth. Patients with rapidly progressive disease, with local symptoms caused by tumor bulk, or with uncontrolled symptoms related to hormone secretion require more aggressive medical or surgical intervention. The somatostatin analogue octreotide may help control hormone secretion and stabilize tumor growth. Patients refractory to octreotide with tumor predominantly in the liver are potential candidates for mechanical ablative techniques, such as hepatic arterial embolization. Radiofrequency ablation and cryosurgical techniques may also be useful, although specific data are limited. Surgical resection of metastatic disease may offer palliative relief of symptoms related to hormone secretion in carefully selected patients. Chemotherapy may be used for palliation when ablative techniques have failed or when significant extrahepatic disease is present. Streptozicin-based combinations remain the first line standard, but major objective responses are less common than had been previously thought. Because of the overall modest success of current chemotherapeutic regimens, patients with advanced disease in need of treatment should be encouraged to enroll in clinical trials testing newer antineoplastic agents or newer treatment strategies.

Fine tangled pili expressed by Haemophilus ducreyi are a novel class of pili.

Haemophilus ducreyi synthesizes fine, tangled pili composed predominantly of a protein whose apparent molecular weight is 24,000 (24K). A hybridoma, 2D8, produced a monoclonal antibody (MAb) that bound to a 24K protein in H. ducreyi strains isolated from diverse geographic locations. A lambda gt11 H. ducreyi library was screened with MAb 2D8. A 3.5-kb chromosomal insert from one reactive plaque was amplified and ligated into the pCRII vector. The recombinant plasmid, designated pHD24, expressed a 24K protein in Escherichia coli INV alpha F that bound MAb 2D8. The coding sequence of the 24K gene was localized by exonuclease III digestion. The insert contained a 570-bp open reading frame, designated ftpA (fine, tangled pili). Translation of ftpA predicted a polypeptide with a molecular weight of 21.1K. The predicted N-terminal amino acid sequence of the polypeptide encoded by ftpA was identical to the N-terminal amino acid sequence of purified pilin and lacked a cleavable signal sequence. Primer extension analysis of ftpA confirmed the lack of a leader peptide. The predicted amino acid sequence lacked homology to known pilin sequences but shared homology with the sequences of E. coli Dps and Treponema pallidum antigen TpF1 or 4D, proteins which associate to form ordered rings. An isogenic pilin mutant, H. ducreyi 35000ftpA::mTn3(Cm), was constructed by shuttle mutagenesis and did not contain pili when examined by electron microscopy. We conclude that H. ducreyi synthesizes fine, tangled pili that are composed of a unique major subunit, which may be exported by a signal sequence independent mechanism.

Haemophilus ducreyi adheres to human keratinocytes.

Haemophilus ducreyi, Moraxella catarrhalis and a non-piliated Escherichia coli K-12 strain were studied for their ability to bind to human keratinocytes in vitro. Epidermal cells isolated from neonatal foreskins were grown to confluence in serum-free keratinocyte media. Probing of the monolayers with anti-cytokeratin antibody showed that 97% of cells were keratinocytes. Bacteria were grown to mid-log phase and seeded onto the monolayers. At various time-points monolayers were washed with PBS to remove non-adherent bacteria, and the monolayers were quantitatively cultured. After 120 min, 15 to 23% of the H. ducreyi inocula bound to the monolayer, while less than 1% of the M. catarrhalis or E. coli controls bound. Wet mounts of fixed monolayers observed with differential interference contrast microscopy confirmed the quantitative data. We conclude that H. ducreyi binds to keratinocytes and that this process may play a role in the initiation of chancroid.